In the summer of 1999, an outbreak of encephalitis in humans that was associated with mosquitoes occurred in New York City (CDC, MMWR, 48, pp. 845-9 (1999); CDC, MMWR, 48, pp. 944-6 (1999); D. S. Asnis et al., Clin Infect Dis, 30, pp. 413-8 (2000)). At approximately the same time, American crows began dying in the Northeastern United States, many in Fairfield County, Conn. Two reports in December of 1999 demonstrated that these outbreaks in birds and humans were actually due to WNV virus transmitted by mosquitoes (R. S. Lanciotti et al., Science, 286, pp. 2333-7 (1999); J. F. Anderson et al., Science, 286, pp. 2331-3 (1999)). It is clear from these reports that WNV was the cause of the 1999 outbreak of fatal encephalitis in the Northeastern United States. This is the first reported appearance of WNV in the Western Hemisphere.
Future outbreaks of WNV in the United States are a new and important public health concern. To date, the only method for preventing WNV infection is spraying large geographic areas with insecticide to kill mosquito vectors. Spraying is difficult, potentially toxic to humans, requires repeated applications and is incompletely effective. There is no known vaccine for use in humans against WNV.
WNV is a member of the family Flaviviridae, genus Flavivirus belonging to the Japanese Encephalitis antigenic complexes of viruses. This sero-complex includes JEV, SLEV, Alfuy, Koutango, Kunjin, Cacipacore, Yaounde, and Murray Valley Encephalitis viruses. This Flaviviridae family also includes the Tick-borne encephalitis virus (TBEV), Dengue virus (including the four strains of: DENV-1, DENV-2, DENV-3, and DENV-4), and the family prototype, Yellow Fever virus (YFV). WNV infections generally have mild symptoms, although infections can be fatal in elderly and immunocompromised patients. Typical symptoms of mild WNV infections include fever, headache, body aches, rash and swollen lymph glands. Severe disease with encephalitis is typically found in elderly patients (D. S. Asnis et al., supra). For the most part, treatment of a subject having a flavivirus infection is a symptomatic treatment, i.e. the general symptoms of a flavivirus infection are treated, such that for initial treatment, mere knowledge of the infection being a flavivirus infection may be sufficient. However, in certain other cases rapid and accurate diagnosis of the specific flavivirus, particularly WNV, is critical such that the most appropriate treatment can be initiated.
Moreover, with respect to the blood suppy (e.g., donor blood to be supplied to patients), and donor organs (e.g., organs to be supplied to patients), there is a need to rapidly determine whether the blood or organs are contaminated by a flavivirus, e.g., determine whether the donor suffers from a flavivirus infection, without needing to know specifically which flavivirus is the source of infection. Conversely, there is also a need for rapid and accurate detection of a specific flavivirus such as WNV since it may be important in some cases to delimit the spread of WNV through the blood supply.
Flavivirus infections are a global public health problem (C. G. Hayes, in The Arboviruses: Epidemiology and Ecology, T. P. Monath, ed., CRC, Boca Raton, Fla., vol. 5, chap. 49 (1989); M. J. Cardosa, Br Med Bull, 54, pp. 395-405 (1998); Z. Hubalek and J. Halouzka, Emerg Infect Dis, 5, pp. 643-50 (1999)) with about half of the flaviviruses causing human diseases. These viruses are normally maintained in a natural cycle between mosquito vectors and birds, where humans and horses are considered dead-end hosts. Birds, including the American crow, Corvus brachyrhynchos, can serve as non-human reservoirs for the virus. In the case of WNV, the virus is transmitted to man by mosquitoes, which in the Northeastern United States are primarily of the genera Culex and Aedes, in particular C. pipiens and A. vexans. 
Flaviviruses are the most significant group of arthropod-transmitted viruses in terms of global morbidity and mortality. An estimated one hundred million cases coupled with the lack of sustained mosquito control measures, has distributed the mosquito vectors of flaviviruses throughout the tropics, subtropics, and some temperate areas. As a result, over half the world's population is at risk for flaviviral infection. Further, modern jet travel and human migration have raised the potential for global spread of these pathogens. Thus, in certain cases, early and rapid detection of a flavivirus infection or of exposure to a flavivirus antigen, without needing to be specific as to which flavivirus, is important. Conversely, it may also be critical to accurately and confidently know the identity of the specific flavivirus causing the infection.
The WNV, like other flaviviruses, is enveloped by host cell membrane and contains the three structural proteins capsid (C), membrane (M), and envelope glycoprotein (E glycoprotein). The E glycoprotein and M proteins are found on the surface of the virion where they are anchored in the membrane. Mature E glycoprotein is glycosylated, whereas M is not, although its precursor, prM, is a glycoprotein. In other flaviviruses, E glycoprotein is the largest structural protein and contains functional domains responsible for cell surface attachment and intraendosomal fusion activities. In some flaviviruses, E glycoprotein has been reported to be a major target of the host immune system during a natural infection.
In general, the flavivirus genome which is replicated in the cytoplasm of the infected cell is a single positive-stranded RNA of approximately 10,500 nucleotides containing short 5′ and 3′ untranslated regions, a single long open reading frame (ORF), a 5′ cap, and a nonpolyadenylated 3′ terminus. The flavivirus genome encodes a single polyprotein which is co- and post-translationally processed by viral and cellular proteases into the three structural proteins, C (capsid), prM/M (premembrane/membrane), and envelope (E glycoprotein) and seven nonstructural proteins, NS1 (nonstructural protein 1), NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (T. J. Chambers et al., Ann Rev Microbiol, 44, pp. 649-88 (1990)).
With respect to post-translational processing of the polyprotein, the sites of proteolytic cleavage in the YFV, which is likely to be predictive of the sites of cleavage in all flaviviruses, have been determined by comparing the nucleotide sequence and the amino terminal sequences of the viral proteins. Subsequent to initial processing of the polyprotein, prM is converted to M during virus release (G. Wengler at al., J Virol, 63, pp. 2521-6 (1989)), and anchored C is processed during virus maturation (Nowak et al., Virology, 156, pp. 127-37 (1987)). In some flaviviruses, the E glycoprotein is the major virus antigen involved in virus neutralization by specific antibodies (Martin D. A., et al. 2002, Clin Diagn Lab Immunol. 9:544-9).
The complete or partial genomes of a number of WNV isolates from the outbreak in the Northeastern United States have been sequenced. The complete sequence of WNV isolated from a dead Chilean flamingo (WN-NY99) at the Bronx Zoo was deposited in GenBank™ (accession number AF196835) (R. S. Lanciotti et al., supra). The genome of a WNV isolate from human victims of the New York outbreak (WNV-NY1999) was sequenced and deposited as GenBank™ accession number AF202541 (X-Y. Jia et al., The Lancet, 354, pp. 1971-2 (1999)). Partial sequences of isolates from two species of mosquito, a crow and a hawk from Connecticut are deposited as GenBank™ accession numbers AF206517-AF206520, respectively (J. F. Anderson et al., supra). Comparative phylogenetic analysis of the NY sequences with previously reported WNV sequences indicated a high degree of sequence similarity between the NY isolates and two isolates from Romania and one from Israel (J. F. Anderson et al., supra; X.-Y. Jia et al., supra; R. S. Lanciotti et al., supra). Furthermore, PCT WO 02/072056 relates to the WNV E glycoprotein and its use in diagnostics of WNV infections. Importantly, the referenced PCT does not at any timerecognize that this antigen is strongly cross-reactive among flaviviruses, such as, JEV, SLEV, and DENV; rather, this PCT publication attempts to advance the proposition that the WNV E glycoprotein is specific for WNV and hence useful to diagnose or detect only WNV or to immunize or vaccinate against only WNV, contrary to the herein inventor's discoveries.
While flaviviruses such as JEV, SLEV, and DENV exhibit similar structural features and components, the individual viruses are significantly different at both the sequence and antigenic levels. Indeed, antigenic distinctions have been used to define four different strains within just the DENV subgroup of the flaviviruses. Infection of an individual with one DENV strain does not provide long-term immunity against the other strains and secondary infections with heterologous strains are becoming increasingly prevalent as multiple strains co-circulate in a geographic area. Such secondary infections indicate that vaccination or prior infection with any one flavivirus may not to provide generalized protection against other flaviviruses.
Serodiagnosis of WNV and other flavivirus infections currently requires a series of enzyme-linked immunosorbant assays (ELISA) and viral plaque reduction neutralization (PRN) tests. Specifically, the recommended assays for the identification of WNV infection of humans are the immunoglobulin M (IgM) antibody capture enzyme linked immunosorbent assay (MAC ELISA), the IgG ELISA (Martin, D. A., 2000, J. Clin. Microbiol. 38:1823-1826; Johnson, A. J., 2000, J. Clin. Microbiol. 38:1827-1831), detection of antibodies in cerebrospinal fluid or serum using a plaque assay (PRN test), isolation of the virus, and RT-PCR. Most public health laboratories in the United States are performing these assays according to protocols recommended by the Centers of Disease Control and Prevention (CDC).
However, the currently available ELISA assays, while not precisely specific for WNV, do not provide for a general diagnostic assay for flavivirus infections (or exposure thereto) with other members of the JEV serogroup (including JEV and SLEV) and DENV because the cross reactivity of the assay to other flaviviruses is unreliable and inconsistent. Further, the currently used ELISA assays according to the CDC do not provide rapid results. Separate assays are currently used to properly and reliably diagnose flavivirus infections other than WNV, such as, JEV, SLEV, and DENV and there is no available assay to reliably, consistently and rapidly detect a flavivirus infection, especially WNV, JEV, SLEV, or DENV. Accordingly, an antigen that is strongly cross-reactive to antibodies against JEV serogroup flaviviruses, especially JEV, SLEV, and DENV for use in a rapid diagnostic assay providing rapid results thereof would be an advance in the art since it would enable a general flavivirus detection assay when knowledge of the specific flavivirus is not necessarily needed. Further, in addition to the current assays that are used to diagnose specific flavivirus infections, antigens for use in new rapid diagnostic assay procedures for the specific diagnosis of a specific flavivirus, such as WNV, that are more accurate, reliable, and sensitive than those currently available would be an important advance in the art.
When rapid, accurate, and sensitive detection of a flavivirus is desired wherein knowledge of the specific flavivirus is not required, an antigen with strong cross-reactivity between flaviviruses is desirable. Further, the antigens currently known in the art lack a sufficient cross-reactivity to allow for reliable, consistent, and accurate testing of a flavivirus infection. One reason limiting the cross-reactivity of current assays in the art, such as, the CDC ELISA assay for the detection of WNV, may relate to the impurity of the antigens used in the assays. The assays used in the art for the detection of WNV and other flaviviruses typically utilize somewhat impure antigens that are contaminated with proportionally high levels of cellular protein and other macromolecules as a result of the purification process. In some cases, the concentration of contaminating protein, such as bovine serum albumin, is greater than the concentration of the antigen being prepared. These impurities can cause a significant reduction in the sensitivity of a given assay (i.e., higher levels of background signals relative to true detection signals) in detecting antibodies against a virus or pathogen of interest from a biological sample. For example, as a control reaction aimed at determining the relative level of background inherent with a given supplied antigen, a separate test of the tissue culture supernatant from which the antigen was obtained may be required. Thus, an antigen that is substantially pure, i.e., one that is not contaminated with unwanted protein or other macromolecules, would be useful for screening for flavivirus infections or exposure thereto since it would provide for a more sensitive diagnosis.
Further, the antigens currently used in the art for the detection of flaviviruses typically are damaged with respect to their three-dimensional structure. For example, damage may occur at specific protein domains or epitopes. Such structural damage is usually introduced during antigen purification and/or isolation wherein the antigen is often treated under harsh and/or destructive conditions that result in damage to an antigen's three-dimensional form. For example, the antigens currently prepared in the art may be treated with the chemical, polyethylene glycol (“PEG”) to help carry out the precipitation of the antigen from solution for the purpose of increasing its concentration. This process can be harmful to a given antigen and may introduce irreversible damage to its structure. Additionally during purification, the antigens can be extracted using acetone. However, acetone extraction can lead to full and/or partial denaturation of the antigen, which, in turn, can result in an antigen having lost its authentic and/or native conformation. Further still, the extent, predictability, reliability, and consistency of cross-reactivity of an antigen is typically greater in the case of an antigen having a authentic and/or native conformation. Thus, it would be useful to have a WNV polypeptide (i.e., antigen) that is of authentic conformation to allow for a stronger, more predictable, more reliable and more consistent cross-reactivity to other flaviviruses, especially, JEV, SLEV and DENV.
In contrast, in situations where cross-reactivity to multiple flaviviruses is undesirable, it would also be an improvement in the art to have available one or more polypeptides (i.e., antigens) that could be used to specifically detect antibodies against a specific flavivirus infection, especially WNV, more accurately, reliably, and rapidly without cross-reactivity with antibodies against other Flaviviruses, such as, for example, JEV, SLEV, or DENV. In other word, in addition to the utility of a polypeptide for the general detection of a flavivirus infection without regard as to which flavivirus, it would also be useful to detect a specific flavivirus infection, such as a WNV infection, using a specific antigen such that the detection of the WNV infection is more reliable, more rapid, and more accurate than currently known methods.
A number of serologic assays are routinely used for laboratory diagnosis of flavivirus infections: IgM antibody capture enzyme immunoassay (MAC-ELISA), indirect IgG ELISA, indirect fluorescent antibody assay (IFAT), hemagglutination inhibition (HIT), and serum dilution cross-species plaque reduction neutralization tests (PRNTs)—each varying markedly in sensitivity, technical difficulty, turn-around time, and clinical utility of the results.
A specific example of a current assay method to detect a flavivirus infection is an assay available from the CDC for the detection of a WNV infection using the COS-1 WNV recombinant antigen (NRA) (Davis, B. S. et al., 2001, J. Virology 75:4040-4047). This antigen can be used in an ELISA procedure to test biological samples for antibodies against WNV. Positive ELISA results are typically confirmed by plaque reduction neutralization (PRN) tests performed in a biosafety level 3 facility. Although this combination of assays is highly sensitive and specific, it requires several days to weeks and specialized facilities to perform the complete panel of tests. For example, the recommended ELISA procedure considered separately takes two to three working days to complete, as overnight incubations are deemed necessary to enhance sensitivity (Martin, D. A., 2000, J. Clin. Microbiol. 38:1823-1826; Johnson, A. J., 2000, J. Clin. Microbiol. 38:1827-1831). Accordingly, the assays currently used in the art to test for WNV and/or other flavivirus infections, such as the COS-1-based assay, are slow and do not provide rapid results. Thus, an assay for determining the presence of a flavivirus infection that is more rapid than those currently available in the art would be useful.
Flavivirus infections occur globally and represent a continued health problem around the world. For example, WNV has recently reached the United States and presently constitutes a growing concern for health officials. Accordingly, surveillance is rapidly becoming more common and widespread. As a result of the increased surveillance, the demand by local health departments and state public health laboratories for critical serologic reagents is far exceeding the supply. Further, the current assays used for WNV detection, such as the CDC-recommended assays mentioned above, are slow and inefficient and do not meet the growing needs of the general health community to rapidly identify infections and to effectively assess WNV epidemiology. Further, current assay methods for detecting WNV are not sufficient to meet growing needs for quicker, more efficient and more sensitive analysis of blood and organ supplies for flavivirus contamination, such as, contamination by WNV and other flavivirus species, including, JEV, SLEV, and DENV. Further still, there exists a need for new diagnostic assays directed to the detection of flavivirus infections in animals to faciliate the handling of infected animal populations or animal populations at-risk of infection. Such diagnostic assays could be in the form of a general flavivirus test wherein knowledge of the specific flavivirus in not required or of a specific flavivirus test, such as a specific test for WNV.
Accordingly, there is a need in the art for methods and kits for improved diagnostic assays for the detection of infections by WNV and other flaviviruses in animals and humans, including JEV, SLEV, and DENV, that are more rapid, cost-effective, efficient and sensitive than the current diagnostic assays available in the art. Further, there is a need for a single assay to enable the general detection of a flavivirus, especially WNV, JEV, SLEV, and DENV, without the requirement of knowing exactly which species is the source of infection. Further still, there is a need in the art for an efficient, sensitive and cost-effective high-throughput diagnostic assay for large-scale detection of flaviviruses to allow for public health control over flaviviral diseases, for example, improving the study of flaviviral-disease epidemiology or improving the ability to analyze blood and organ supplies. In addition to the need for a general flavivirus detection method, there also exists a need for improved methods to enable the rapid and reliable detection of a specific flavivirus, such as WNV and DENV, without substantial cross-reaction to other flaviviruses. Moreover, with respect to DENV, there exists a further need for a method to reliably and rapidly descriminate between the different strains of DENV, including DENV-1, DENV-2, DENV-3, and DENV-4. Further still, a need exists for a method that is capable also of discriminating reliably between a recent and/or current flavivirus infection and a previous infection of a flavivirus in a manner that is either specific or nonspecific as to the flavivirus of the infection.